1. Field of the Invention
The present invention relates to a laser scanning optical system and a laser scaning optical apparatus for performing scanning of laser beam, used in various fields, for example in biology, in medicine, in semiconductor engineering, as a fluorescence microscope, an optical writing apparatus, and as an IC repair apparatus.
2. Related Background Art
If an ordinary fluorescence microscope is used to observe a sample having a three-dimensional thickness, defocused images outside the depth of focus are superimposed on an image formed on the focal plane. This globally lowers the contrast of microscopic image, which makes determination of fluorescence intensity difficult. Approaches to deal with this problem will be described in the following.
For example, in a conventional confocal laser scanning fluorescence microscope, a laser beam emitted from a laser is expanded in diameter of its ray bundle by a beam expander and thereafter passes through a dichroic mirror. The laser oscillates the laser beam at a wavelength corresponding to a peak wavelength in an absorption spectrum of a fluorescent dye used in labeling of a sample. The beam expander is composed of two convex lenses. The dichroic mirror is so formed as to have a high reflectivity for optical components in a predetermined wavelength range including a fluorescence emitted from the fluorescent dye and a high transmittance for the oscillation wavelength of the laser beam.
The laser beam is bent in the direction perpendicular to the optical axis by an X-Y scanner and is focused by an imaging lens to form an optical spot on a front image plane of an objective lens, and thereafter the objective lens converges the optical spot to the diffraction limit to form a converged optical spot inside the sample. The X-Y scanner changes the traveling direction of laser beam within a predetermined angular range to scan in two orthogonal directions on a plane. Further, the objective lens or a stage on which the sample is set moves in parallel with the optical axis. Thus, the optical spot of the laser beam three-dimensionally scans the inside of sample by parallel scanning to the optical axis in addition to the two-dimensional scanning perpendicular to the optical axis, such as raster scanning. The sample is, for example, an organism sample labeled with a fluorescent dye, which is excited by the optical spot of the laser beam.
A fluorescence diverging out of the sample is collected by the objective lens and thereafter advances backward through the optical path, through which the laser beam has passed. The fluorescence outgoing from the X-Y scanner is reflected by the dichroic mirror in the direction perpendicular to the optical axis and thereafter is focused by a collimator lens to form an image thereof in a pinhole in a confocal pinhole plate. The fluorescence outgoing from the confocal pinhole plate is separated from fluorescence components emitted from positions before and after the optical spot inside the sample and is received by a PMT (Photo Multiplier Tube).
The PMT photoelectrically converts the fluorescence into an electric signal corresponding to the light intensity thereof and outputs it. The electric signal output from the PMT is stored as image data in a memory in an image reading apparatus in synchronization with a scanning signal of the X-Y scanner. A three-dimensional microscopic image of the sample is obtained by processing the image data in correspondence with the scanning signal by ordinary procedure.
The conventional confocal laser scanning fluorescence microscope as described above employs such an arrangement that ideally a point light source and a point photodetector are located at positions conjugate with a point inside the sample whereby the laser beam forms an optical spot having a reduced focal depth. Also, the pinhole is located on the photodetector side so as to remove the fluorescence components emitted from positions before and after the optical spot inside the sample. This can eliminate almost all defocused images except for an image on the focal plane. Accordingly, only the image near the focal plane inside the sample is obtained as a microscopic image.
The prior art on such a confocal laser scanning fluorescence microscope is described in detail, for example, in "Japanese Laid-open Patent Application No. 2-247605".
Also, a conventional two-photon absorption excitation type laser scanning fluorescence microscope employs a laser oscillating a laser beam as a pulse having a very short time duration, in which the laser beam forms an optical spot having a high energy density and the optical spot three-dimensionally scans the inside of a sample in the same manner as in the confocal laser scanning fluorescence microscope as described previously. Because of the arrangement, a fluorescence due to excitation based on two-photon absorption appears only from a point where the optical spot is located inside the sample but no fluorescence due to excitation based on two-photon absorption appears from other portions. Therefore, there appears no defocused image other than one on the focal plane, which improves the contrast of the microscopic image.
The prior art on such a two-photon absorption excitation type laser scanning fluorescence microscope is described in detail for example in references "Science, vol. 248, pp. 73-76, 6 Apr. 1990" and "U.S. Pat. No. 5,034,613, 1991".
Further, conventional general laser scanning fluorescence microscopes employ an axicon prism replacing the objective lens, by which laser beams interfere with each other on the optical axis to be converted into a bundle of rays having a focal depth as long as the thickness of sample, i.e., into a so-called Bessel beam and through which the Bessel beam three-dimensionally scans the inside of sample in the same manner as in the confocal laser scanning fluorescence microscope as described previously. Therefore, an image within the focal depth will never be unfocused, thus obtaining a microscopic image as a two-dimensional projection of a three-dimensional image. In another arrangement a laser beam is guided through an aperture having an annular opening to be shaped in a cylindrical bundle of rays to enter an objective lens, the laser beam forms a Bessel beam having a depth of focus as long as the thickness of the sample, and then the Bessel beam three-dimensionally scans the inside of sample in the same manner as in the confocal laser scanning fluorescence microscope as described previously. Thus, an image within the focal depth will never be unfocused, obtaining a microscopic image as two-dimensional projection of a three-dimensional image.
A conventional optical converting unit for producing a Bessel beam is so arranged that an aperture having an annular opening portion is set on the front focal plane of a convex lens. If a laser beam passes as a beam of parallel rays through the opening portion in the aperture, a diffracted light is produced as a bundle of rays having an annular cross-sectional intensity distribution perpendicular to the optical axis. The diffraction light having passed through the convex lens advances as a plane wave refracted at a constant angle relative to the optical axis and thereafter forms a conical wavefront in axial symmetry with respect to the optical axis at the rear focus of the convex lens. Thus, beams of the diffracted light mutually interfere in the entire region where the wavefront exists near the optical axis, so that a Bessel beam is produced with an intensity enhanced by constructive interference.
In an intensity distribution of the Bessel beam in a cross section perpendicular to the optical axis, a thin linear center beam with strong intensity with the interference region near the optical axis exists almost constantly. On the other hand, concentric cylindrical higher-order diffraction beams with small intensity are present at positions away from the optical axis. It is thus understood that the Bessel beam has a high resolution and a long focal depth.
The prior art on such an annular illumination optical system using the aperture in the laser scanning fluorescence microscope is described in detail, for example, in "Optics, vol. 21, no. 7, pp. 489-497, July 1992". Also, the prior art on production of the Bessel beam by the axicon prism is described in detail, for example, in "Laser Microscope Research Group, the tenth lecture papers, pp. 22-29, November 1992". Further, the prior art on the annular illumination optical system using the axicon prism is described in detail for example in "U.S. Pat. No. 4,887,592, 1989".
However, the conventional confocal laser scanning fluorescence microscope as described above shields the fluorescence components emitted from positions before and after the optical spot inside the sample by locating a pinhole on the photodetector side. This extremely lowers the reception efficiency of fluorescence in the photodetector, which results in a problem of further reducing the intensity of originally weak fluorescence.
Also, in case of the two-photon absorption excitation type laser scanning fluorescence microscope as described above, the fluorescence appears only from an optical spot inside the sample and therefore in order to obtain a three-dimensional microscopic image, the optical spot is scanned in parallel with the optical axis inside the sample in addition to the two-dimensional scanning perpendicular to the optical axis. This requires a long time as the scanning time of the optical spot. In this time a sample of an organism having activity could move, resulting in failing to obtain a correct three-dimensional microscopic image. In addition, there is a problem that a discoloration state or an exhaustion state of fluorescent dye becomes locally different.
Further, the conventional general laser scanning fluorescence microscopes as described above employ the axicon prism replacing the objective lens whereby the laser beam forms a Bessel beam having a focal depth as long as the thickness of sample. Thus, the axicon prism has no imaging function and a state of convergence is not good for the bundle of rays irradiated onto the sample, which results in a problem that the resolution is low in the plane perpendicular to the optical axis of axicon prism. In another case, a laser beam is guided through an aperture having an annular opening to form a bundle of rays having an annular cross section intensity perpendicular to the optical axis and the bundle is made incident into the objective lens, whereby the laser beam forms a Bessel beam having a focal depth as long as the thickness of the sample. Even if the laser beam has a cross-sectional intensity distribution based on an approximately Gaussian distribution, a bundle of rays with peak intensity is shielded by the disc shielding portion in the aperture, which causes a problem that the utilization factor of the laser beam irradiated onto the sample is extremely lowered. Also, the bundle of rays or the optical spot of the laser beam having a large focal depth as described above forms annular beams of the higher-order diffraction beams around a linear beam. This causes a problem that when such a bundle of rays or the optical spot scans the inside of sample, a lot of false signals are generated.
Therefore, the present invention has been accomplished in view of the above problems and an object of the present invention is to provide a laser scanning optical system and a laser scanning optical apparatus which can perform scanning of laser beam with a higher energy utilization factor, a higher resolution and a longer focal depth within a shorter time than the prior art apparatus did.